Cardiac tissue engineered from newborn rat heart cells reveals fundamental sex-based differences in how cellular architecture relates to contractile performance. Male-derived cardiac monolayers demonstrated distinct sarcomere organization and tissue alignment patterns compared to female samples, with corresponding variations in contraction force and electrical conduction velocity. These structural disparities emerged even before hormonal influences, suggesting intrinsic genetic programming drives early cardiac development differences between sexes. The findings challenge the prevailing assumption that sex-based cardiac differences only manifest after puberty through hormonal modulation. This research carries significant implications for regenerative cardiology and drug development, where most preclinical models use male cells exclusively. Understanding these fundamental differences could explain why women experience different cardiovascular disease patterns and drug responses throughout life. The work also suggests that tissue engineering approaches for heart repair may need sex-specific optimization strategies. However, the study's limitation to neonatal rat cells means human applications remain speculative. Still, this represents a paradigm shift toward recognizing sex as a biological variable from the earliest stages of cardiac development, potentially informing more personalized approaches to treating heart disease across the lifespan.